Increasing evidence indicates that hepatocyte growth factor (HGF) plays an important role in uterine development and pathological conditions such as endometriosis and carcinoma. Proteolytic conversion of single chain HGF to active heterodimeric HGF in the extracellular milieu is a critical limiting step in HGF- mediated signal transduction, however, the mechanisms controlling HGF activation in the uterus remain unexplored. The long-term goal of the current proposal is to determine the factors involved in the activation of HGF in the uterus, and elucidate the underlying regulatory mechanisms. HGF activator (HGFA) is a serine protease and is most potent in processing HGF activation in vivo, but no information is available regarding its expression in the uterus. The specific aim of this application is to examine the expression and steroid hormonal regulation of the HGF/c-Met regulatory system including HGFA and its inhibitors HAI-1 and HAI-2 in the uterus, and characterize the role of HGFA in the activation of HGF/c-Met signaling. To accomplish this goal, this study will use both in vivo and in vitro models to examine the expression of HGFA, HAI-1, HAI-2, HGF and c-Met in the mouse uterus as well as their regulation by estrogen and progesterone. The role of HGFA in HGF activation will be examined by correlating HGFA expression with HGF activation, c-Met phosphorylation and endometrial epithelial and stromal cell proliferation. Recombinant HGFA, neutralizing antibodies for HGFA and HGF will be utilized during in vitro study to confirm the specific effect of HGFA through HGF activation. Methods used in this proposal include endometrial stromal and epithelial cell culture, Western analysis, Immunohistochemistry, Real-time RT-PCR, In situ hybridization, HGF activation assay and Cell proliferation assay. Data obtained from these experiments will provide novel information on the cellular localization, regulation and function of HGFA in uterine physiology. Further more, this research will provide groundwork for thorough investigation on the molecular mechanisms controlling HGF activation in the uterus, and may even lead to the development of new strategies in treating uterine diseases characterized by abnormal expression and function of HGF, such as endometriosis. The findings from the proposed studies will provide novel information on the regulation of the uterine HGF system and may lead to the development of new therapies to target endometrial pathophysiologies such as endometriosis. [unreadable] [unreadable] [unreadable]